A critical step in the replication of human immunodeficiency virus (HIV) is the formation of an initiation complex composed of the HIV genome and a cellular tRNA. This binary RNA complex in conjunction with viral encoded proteins including, reverse transcriptase (RT) and the nucleocapsid protein (NCp7), mediates the elongation of the primer tRNA to form a short DNA intermediate (minus-strand stop DNA) from R-U5 sequences of the viral RNA. The cellular tRNALys, 3 is selectively used in this early stage of HIV-1 replication and two complementary sequences within the retrovirus R-U5 have been shown to be important in early replication steps. An 18 nucleotide sequence within the virus genome termed the primer binding site (PBS) is complementary to the 3' terminus of tRNALYS, 3 and an adenine rich sequence (A-loop) upstream of the PBS is complementary to tRNALYS, 3 anti-codon. Formation of the initiation complex requires the unfolding of both the viral template and tRNA primer and the formation of a new RNA structure. While the role of RT in this initial stage of HIV replication has been extensive- ly investigated little is known about the structural requirements for the initiation complex. The goal of this project will be to provide the structural information necessary to first formulate a plausible tertiary structure model of the initiation complex and second to functionally test this model by site directed mutagenesis of critical structural elements. The specific aims of this proposal are 1) to determine the secondary structure of the tRNALys, 3-HIV-1 replication initiation complex and alternative initiation complexes formed with tRNAHis and tRNAMet; 2) to establish selective tertiary structure constraints by intra- and inter- molecular crosslinking of the tRNALys,3-HIV-1 initiation complex and alternative initiation complexes formed with tRNAHis and tRNAMet; 3) to develop an in vitro selection strategy to identify novel tRNA-HIV priming complexes. These studies will provide the first detailed analysis of the structural features of the RNA:RNA interactions required for initiation of HIV-1 reverse transcription. This critical step in the HIV life-cycle is an appealing target for drug intervention and the structural information from our studies will provide the basis for the development of such compounds.